Determining the 3D structure of a specimen is of significant importance in various fields, e.g., biological and materials research. Block face imaging inside the chamber of a scanning electron microscope (SEM) is one way of determining the make-up of such structures. As is known, a higher resolution 2D image can sometimes be obtained in a SEM if the electron landing energy is reduced by applying a negative retardation voltage to the specimen. Accordingly, the application of a retardation field to the specimen is a growing trend in electron microscopy and is being offered routinely by JEOL, PEI and Hitachi on select SEM models. The retardation voltage is applied to the specimen being imaged and this voltage is configurable by the user through a dedicated user interface. The retardation voltage is typically less than the column high tension (energized voltage of electrons leaving the pole piece of the column). The landing energy of electrons on the specimen is close to the difference between these two voltages. When detecting backscattered electrons the associated electric field has a double benefit as the re-energizing of the backscattered electrons stimulates a stronger response by the detector. A common configuration is for the retardation voltage to be applied to the specimen holder which is electrically isolated from the SEM chamber, which remains at ground potential throughout. The retardation field reduces the landing energy of the electrons. However, since the field can never be entirely symmetrical to the pole piece of the microscope, the field typically causes some shift in the position of the beam, in the focus and in the astigmatism of the focused probe.
Utilizing a microtome within the chamber of an SEM making use of a retardation field to take sequential slices of the specimen for block face imaging presents the problem that the knife blade of the microtome, or knife blade holding structure will interfere with the retardation field. This will cause image shift effects between sequential slice images which will degrade the alignment and quality of a 3D “stack” of images.